Ion channels and calcium regulation in vascular SMC function

描述（由申请人提供）：我们的长期目标是确定不同Ca2+进入途径在血管平滑肌细胞（SMC）功能中的分子机制和生理作用。这一建议是我们对血管SMC中储存操作通道（SOC）和Ca2+进入（SOCE）的成功研究的逻辑延续。最近，我们发现同一个Orai1基因可以编码具有截然不同选择性的通道，adar1介导的RNA编辑可能是不同细胞类型中Orai1特性调控的分子机制。我们在原发性血管SMC的Orai1 mRNA中发现了假定的RNA编辑位点，并证明单点突变（模仿RNA编辑事件）可以将Ca2+选择性CRAC转化为cat-SOC通道。我们还获得了ipla2b依赖性的Orai1激活可能对原发性血管SMC的增殖和迁移至关重要的证据。我们的初步研究为我们的新建议提供了坚实的概念和实验基础，以解决长期以来关于血管SMC中cat-SOC通道的分子特性的争议，确定SMCmigration中新的Ca2+入口机制，并建立新的分子机制和治疗心血管疾病的靶点。我们的中心假设是rna编辑的Orai1编码cat-SOC通道，并在血管SMC迁移中发挥重要作用。我们建议使用我们的综合方法进行深入的研究，包括初级血管SMC和模型细胞系中单个分子和信号级联的高级分子，生化，成像，电生理和功能表征。所有的方法和方法都在PI的实验室中得到了成功的应用。广泛的初步数据充分证明了拟议研究的可行性。我们建议：目标1。为了确定Orai1的rna编辑的分子机制和后果。我们将验证Orai1是adar1依赖性RNA编辑的生物学靶点的假设，将表征可以改变Orai1编码通道的选择性、糖基化和其他生物物理特性的RNA编辑事件，并确定单个Orai1亚基的编辑如何影响Orai1四聚体的特性。目标2。探讨原生猫-有机碳通道在原发性血管SMC中的分子结构。我们将验证RNA编辑的Orai1编码天然猫- soc通道的假设，并将表征adar1介导的Orai1在原发性血管SMC中的RNA编辑和去糖基化。目标3。为了确定Orai1参与SMC迁移的新作用和分子机制：我们将确定iPLA2b和Orai1在SMC迁移中的作用和空间分布，并验证Orai1介导的Ca2+进入参与局灶粘连形成和成熟，和/或使SMC迁移的力产生的假设。

DESCRIPTION (provided by applicant): Our long term goal is to define molecular mechanism and physiological role of different Ca2+ entry pathways in vascular smooth muscle cell (SMC) function. This proposal is a logical continuation of our successful studies of the store-operated channels (SOC) and Ca2+ entry (SOCE) in vascular SMC. Recently we discovered that the same Orai1 gene can encode channels with profoundly different selectivity, and ADAR1-mediated RNA editing can be a molecular mechanism for versification of Orai1 properties in different cell types. We identified putative RNA editing sites in Orai1 mRNA from primary vascular SMC, and demonstrated that single point mutation (that mimics RNA editing event) can transform Ca2+ selective CRAC into cat-SOC channel. We also obtained evidence that iPLA2b-dependent activation of Orai1 may be crucial for proliferation and migration of primary vascular SMC. Our preliminary studies provided solid conceptual and experimental foundation for our new proposal that is posed to resolve the long lasting controversy about the molecular identity of cat-SOC channels in vascular SMC, identifynew Ca2+ entrymechanisminvolved in SMCmigration, andestablish new molecular mechanisms and targets for treatment of cardiovascular diseases. Our central hypothesis is that RNA-edited Orai1 encodes cat-SOC channels and plays important role in vascular SMC migration. We propose in-depth studies using our integrative approach that involves advanced molecular, biochemical, imaging, electrophysiological and functional characterization of individual molecules and signaling cascades in primary vascular SMC and model cell lines. All approaches and methods are successfully used in the PI's lab. The feasibility of proposed studies is fully justified by extensive preliminary data. We propose: Aim 1. To determine the molecular mechanism and consequences of RNA-editing of Orai1.. We will test hypothesis that Orai1 is a biological target for ADAR1-dependent RNA editing, will characterize RNA editing events that can change selectivity, glycosylation and other biophysical properties of Orai1-encoded channels, and determine how editing of individual Orai1 subunits affect the properties of Orai1 tetramers. Aim 2. To identify molecular organization of native cat-SOC channel in primary vascular SMC. We will test hypothesis that RNA-edited Orai1 encodes native cat-SOC channel and will characterize ADAR1-mediated RNA editing and de-glycosylation of Orai1 in primary vascular SMC. Aim 3. To establish the novel role and molecular mechanism of Orai1 involvement in migration of SMC: We will determine the role and spatial distribution of iPLA2b and Orai1 in migrating SMC and test the hypothesis that Orai1-mediated Ca2+ entry is involved in focal adhesion formation and maturation, and/or force production that enables migration of SMC. PUBLIC HEALTH RELEVANCE: Our long term goal is to define the role of specific ion channels in calcium homeostasis which regulates vascular smooth muscle cell (SMC) function. The goals of this proposal are to determine the role of Orai1 (a specific plasma membrane protein) in encoding native store-operated calcium influx channels, and to establish their physiological role in SMC migration. The feasibility of these studies is fully supported by extensive preliminary data and advanced expertise of PI's lab.